Figure 1.
In vivo kinetics of nuclear translocation of MR in COS-7 cells.
COS-7 cells transfected with GFP-MR were treated with 10−8 M aldosterone (Aldo), 10−8 M aldosterone +10−6 M torasemide (Aldo+ Tora), 10−8 M aldosterone +10−6 M spironolactone (Aldo+Spiro), 10−6 M spironolactone (Spiro) or 10−6 M torasemide (Tora), starting at time 0. Individual points represent the average percentage fluorescent intensity of the nucleus vs. total cellular fluorescence (Fn/Ft) measured in individual cells over the indicated period of time (± SE, n = 16). Data points were fitted to a sigmoid curve.
Figure 2.
Effect of Torasemide on ligand-dependent transactivation activity of MR in H9C2-MR cells.
A: The MMTV promoter contains Hormone Response Elements (HRE); the promoter sequence was fused to the Luciferase coding sequence. This construct was transfected into H9C2-MR cells. Upon binding of the aldosterone-MR complexes on HRE, luciferase is transcribed and light emission is enhanced, assessing MR transactivation activity. In the presence of an MR antagonist, binding of aldosterone to MR is prevented and luciferase expression is abolished. B: 10−8 M aldosterone (Aldo) increased MR transactivation activity (luciferase activity) which was prevented by a 100-fold excess of the MR antagonist spironolactone (Spiro; 10−6 M) but not by the GR antagonist RU 38486 (RU 38486; 10−6 M). Each antagonist alone has no effect. Mean ± SEM (n = 8). * p<0.05 vs control (Ctrl); # p<0.05 vs aldosterone. C: 10−8 M aldosterone (Aldo) increased MR transactivation activity, which was fully inhibited by the MR antagonist spironolactone (S) at 10-6 M. Torasemide (T) has a slight antagonist effect independent of its concentration. Mean ± SEM (n = 4). *p<0.05 vs control (Ctrl); # p<0.05 vs aldosterone.
Figure 3.
Torasemide does not act as a MR antagonist for the regulation of endogenous genes in H9C2-MR cells in the presence of 10−8 M aldosterone.
10−8 M aldosterone (Aldo) increased expression of the aldosterone-targets genes Sgk-1, PAI-1, Orosomucoid-1, Rgs-2, Serpina-3 and Tenascin-X. Addition of increasing doses of spironolactone (A+S) inhibited aldosterone-induced gene expression. In contrast, increasing concentrations of torasemide (A+T) had no antagonistic effect. Mean ± SEM (n = 4). *p<0.05 vs control (Ctrl); # p<0.05 vs aldosterone.
Figure 4.
Torasemide does not act as a MR antagonist for the regulation of endogenous genes in H9C2-MR cells in the presence of 10−9 M aldosterone.
10−9 M aldosterone (Aldo) increased expression of the aldosterone-targets genes Sgk-1, PAI-1, Orosomucoid-1, Rgs-2, Serpina-3 and Tenascin-X. Addition of increasing doses of spironolactone (A+S) inhibited aldosterone-induced gene expression. In contrast, increasing concentrations of torasemide (A+T) had no antagonistic effect. Mean ± SEM (n = 4). *p<0.05 vs control (Ctrl); # p<0.05 vs aldosterone.